RESUMO
Transcriptional activation by, and therefore the physiologic impact of, activated tyrosine-phosphorylated STATs (signal transducers and activators of transcription) may be negatively regulated by proteins termed PIAS (protein inhibitors of activated stats), as shown by previous experiments with mammalian cells in culture. Here, by using the genetic modifications in Drosophila, we demonstrate the in vivo functional interaction of the Drosophila homologues stat92E and a Drosophila PIAS gene (dpias). To this end we use a LOF allele and conditionally overexpressed dpias in JAK-STAT pathway mutant backgrounds. We conclude that the correct dpias/stat92E ratio is crucial for blood cell and eye development.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas de Insetos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas/fisiologia , Transativadores/antagonistas & inibidores , Alelos , Animais , Proteínas de Transporte/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Olho/embriologia , Olho/ultraestrutura , Anormalidades do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Janus Quinases , Morfogênese , Mutagênese Insercional , Neoplasias Experimentais/genética , Fosforilação , Proteínas Inibidoras de STAT Ativados , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Recombinação Genética , Fatores de Transcrição STAT , Transdução de Sinais , Fatores de TranscriçãoRESUMO
Tissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype was attributed to premature nuclear translocation of the PERIOD/TIMELESS heterodimer. Reducing SGG/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreased TIMELESS phosphorylation, and it was found that GSK-3 beta specifically phosphorylates TIMELESS in vitro. Overexpression of sgg in vivo converts hypophosphorylated TIMELESS to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated TIMELESS of wild-type flies. Our results indicate a role for SGG/GSK-3 in TIMELESS phosphorylation and in the regulated nuclear translocation of the PERIOD/TIMELESS heterodimer.
Assuntos
Relógios Biológicos/fisiologia , Ritmo Circadiano/fisiologia , Proteínas de Drosophila , Drosophila melanogaster/fisiologia , Quinase 3 da Glicogênio Sintase , Proteínas de Insetos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Relógios Biológicos/genética , Núcleo Celular/metabolismo , Ritmo Circadiano/genética , Dimerização , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Immunoblotting , Proteínas de Insetos/genética , Microscopia de Fluorescência , Atividade Motora/genética , Atividade Motora/fisiologia , Proteínas Circadianas Period , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA/genética , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We describe a new experimental technique that allows for a tissue-specific reduction of gene activity in the Drosophila nervous system. On the basis of the observation that certain gene functions can be ubiquitously blocked by injecting double-stranded RNA into Drosophila embryos, we employed a method to interfere with an individual gene function permanently in a predetermined cell type. This was achieved by the formation of an inverted-repeat RNA sequence in the tissue of interest under control of the GAL4/UAS binary expression system. As an example, we show that inverted-repeat-mediated interference with the period gene produces a hypomorphic period phenotype. A selective decrease of period RNA appears to be a component of the cellular response.
Assuntos
Comportamento Animal , Ritmo Circadiano/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica/genética , Proteínas Nucleares/genética , RNA de Cadeia Dupla/farmacologia , Sequências Repetitivas de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae , Animais , Animais Geneticamente Modificados , Inversão Cromossômica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Proteínas Fúngicas/genética , Proteínas de Insetos/genética , Proteínas de Insetos/fisiologia , Microinjeções , Proteínas Nucleares/fisiologia , Especificidade de Órgãos , Proteínas Circadianas Period , Células Fotorreceptoras de Invertebrados , Regiões Promotoras Genéticas , RNA Antissenso/farmacologia , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Rodopsina/genética , Fatores de Transcrição/genéticaRESUMO
The tumor suppressor gene p53 in mammalian cells plays a critical role in safeguarding the integrity of genome. It functions as a sequence-specific transcription factor. Upon activation by a variety of cellular stresses, p53 transactivates downstream target genes, through which it regulates cell cycle and apoptosis. However, little is known about p53 in invertebrates. Here we report the identification and characterization of a Drosophila p53 homologue gene, dp53. dp53 encodes a 385-amino acid protein with significant homology to human p53 (hp53) in the region of the DNA-binding domain, and to a lesser extent the tetramerization domain. Purified dp53 DNA-binding domain protein was shown to bind to the consensus hp53-binding site by gel mobility analysis. In transient transfection assays, expression of dp53 in Schneider cells transcriptionally activated promoters that contained consensus hp53-responsive elements. Moreover, a mutant dp53 (Arg-155 to His-155), like its hp53 counterpart mutant, exerted a dominant-negative effect on transactivation. Ectopic expression of dp53 in Drosophila eye disk caused cell death and led to a rough eye phenotype. dp53 is expressed throughout the development of Drosophila with highest expression levels in early embryogenesis, which has a maternal component. Consistent with this, dp53 RNA levels were high in the nurse cells of the ovary. It appears that p53 is structurally and functionally conserved from flies to mammals. Drosophila will provide a useful genetic system to the further study of the p53 network.
Assuntos
Drosophila melanogaster , Genes de Insetos , Genes p53 , Proteínas de Insetos/genética , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Animais , Apoptose , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Análise de Sequência , Homologia de Sequência de AminoácidosRESUMO
Recent studies suggest that members of the cadherin family of homophilic cell adhesion molecules play an important role in the formation and stabilization of the complex neural circuitry of the brain.
